We will continue to explore the molecular basis for X chromosome dosage compensation as follows: 1) We will examine DNA methylation of Factor IX and OTC loci; We will look for differences in methylation of these genes on active and inactive X in nonexpressing as well as expressing tissues, so as to determine the relationship between DNA methylation, transcription and dosage compensation at these loci. 2) We will continue studies of chorionic villi and derivative mouse-human hybrids, to further characterize the incomplete dosage compensation in this tissue. 3) We will examine DNA methylation of X linked genes in the marsupial genome to discover the basis for the incomplete dosage compensation observed in this mammal. We will look for presence of CpG clusters at Hprt and G6pd loci, and compare the state of methylation and chromatin of loci that are expressed, and those that are silent or partially expressed. 4) We will continue studies of the replication patterns in active, inactive and reactivated X chromosomes using mouse-human hybrids having one X chromosome and not the other, or both active and inactive X. Using photodegradation of BUdR substituted DNA, and multiple X-linked probes, we will determine the extent of differences (if any) in the replication of homologous loci on active, inactive and reactivated X. 5) We will look for insights into the initial steps in the process from studies of cells with incomplete dosage compensation. 6) We will utilize the cloned probes for centric heterochromatin from chromosome 6 and the X to characterize the relevant heterochromatin with respect to time of replication, and their potential as cytological markers in studies of arrangement of chromosomes in the interphase nucleus and as a means to "karyotype" the interphase cell. 7) We will continue studies of females for X linked mutations to characterize in vivo selection in mosaic cell populations.